Biosensors are commonly used to perform kinetic studies of complex molecular interactions such as those between drug-target, hormone-receptor, enzyme-substrate and antigen-antibody. The biosensors are typically in a flow injection-based fluidic system wherein one or more sensing regions are housed within a flow cell conduit of the fluidic system. The fluidic system further includes one or more flow channel conduits that direct fluid flow to the sensing regions in the flow cell conduit. The sensing region provides surfaces that support immobilized molecules referred to generally as “ligands.” The ligands are potential binding partners for molecules known as “analytes” which are present in fluids that are directed to the sensing region of the flow cell conduit via the flow channel conduit. Typically one member of an affinity pair, the ligand, is immobilized onto a surface in the sensing region while the second member, the analyte, is exposed to this ligand-coated surface for sufficient time to form analyte-ligand complexes at the sensing region. The accumulation of the resulting affinity complexes at the sensing region is detected by a label-free detection method selected from the group consisting of evanescent filed-based optical refractometers, surface plasmon resonance (SPR), optical interferometers, waveguides, diffraction gratings, photonic crystal waveguide arrays, and gravimetric microbalances based on frequency dampening of piezoelectric substrates. The biosensor response is then plotted in real-time and is referred to as a response curve or binding response curve. In order to accurately determine the affinity complex interaction parameters (binding affinity constants, association constants, dissociation constants, diffusion coefficients, etc), the ligand is generally exposed to multiple analyte concentrations.
In WO2012/087840 A1, we described an injection method that permitted a continuum of smoothly changing concentration to be tested through a single injection of sample having a single, starting analyte concentration. In this method, the multiple analyte concentrations are generated en route to the flow cell due to the sample undergoing a mathematically-defined dispersion event thereby producing a well-characterized concentration gradient profile. In order to fit within this mathematical model, the physical injection conditions must be performed in accordance with certain rules relative to, for example, tube length, tube diameter, and flow rate. In one embodiment, Taylor's theory of dispersion provides the basis for the mathematically-defined dispersion event such that the physical injection conditions must be consistent with and comply with the assumptions and limits defined by Taylor dispersion theory. Not only did this method allow for an accurate determination of analyte concentration at any injection time (a feature not present in the prior art single injection methods utilizing uncharacterized concentration gradients), it also provided the ability to determine the diffusion coefficient of the analyte. In more practical terms, the previous dispersion method provides significantly greater throughput in terms of sample number and faster run times, and also significantly extends biophysical characterization in a label-free biosensor.
There are a few limitations on the previous dispersion method. First, the physical conditions under which the injection can occur are limited to those that comply with dispersion model utilized. Under Taylor's model, the flow rate must be low relative to the volume and diameter of the flow channel and can't exceed certain levels without producing a dispersion event that is not consistent with Taylor theory. For practical purposes, the low flow rate equates to a longer injection time thereby decreasing throughput. Thus, an improvement to the method that permits the use of higher flow rates while not departing from the Taylor model is desired.
Second, the previous dispersion method did not account for analyte interactions with the tubing wall en route to the flow cell. When present, these interactions reduce the average velocity of the analyte population relative to the solvent (liquid carrying the analyte) front thereby effecting the residence time of the analyte. By not accounting for this retention time, the dispersion model fails to accurately reflect the gradient profile as the injection progresses. Furthermore the retention of analyte at the wall can provide added functionality such as chromatographic separation and significant differentiation over existing technology. The improved dispersion method described herein addresses these concerns and provides added functionality.